1. Field of the Invention
The present invention relates generally to the fields of developmental biology and molecular biology. More specifically, the present invention relates to the cloning and expression of a novel gene, bves, whose nucleic acid sequence encodes a blood vessel/epicardial substance, Bves, protein involved in cardiovascular or skeletal muscle diseases.
2. Description of the Related Art
Diverse cell types including myocytes, endocardial endothelial cells (hereafter referred to as endocardium), fibroblasts, epicardium, vascular endothelium and vascular smooth muscle make up the heart (Fishman and Chien, 1997). These cells populate the heart at different times and have diverse. origins. Myocytes and endocardium arise from epithelia located in the anterior lateral mesoderm of the embryo and are the first cells to comprise the heart (DeHann, 1965; Coffin and Poole, 1988; Sater and Jacobson, 1989; Gonzalez-Sanchez and Bader, 1990; Fishman and Chien 1997). After establishment of this two-layered epithelial tube, additional migratory mesenchymal cells move into the forming heart. Among these are neural crest cells that are targeted to specific regions of the outflow tract and cardiac skeleton where they differentiate into connective tissue and smooth muscle (Kirby et al, 1978; Kirby and Waldo, 1990) and cells of the proepicardial organ that will differentiate into at least four different cell types.
The proepicardial organ is a transitory epithelial structure located at the septum transversum (Manasek 1968). At stage 15 in the chick and 9.5 dpc in the mouse, the proepicardial organ is situated at the root of the sinoatrium on its dorsal surface (Manasek, 1968; Komiyama et al, 1987). Strands of epithelium begin to extend over the heart forming the future epicardium while the future pericardium is formed as the proepicardial strands extend over the pericardial cavity (Manasek, 1968; Manner, 1993). A subpopulation of epicardial cells begins to delaminate and migrate into the subepicardial connective tissue and myocardium (Manasek, 1968; Viragh and Challice 1981; Hiruma and Hirakow 1989; Manner 1993). These cells will differentiate into fibroblasts, vascular endothelium and vascular smooth muscle (Mikawa and Gourdie, 1996; Dettman et al, 1998). While clonal analysis has shown that divergence of these lineages likely has already taken place in the proepicardial organ (Mikawa and Gourdie, 1996; Dettman et al, 1998), the timing of commitment, differentiation and patterning of endothelial and smooth muscle cells is unresolved.
Development of the intracardiac circulation is unique in vasculogenesis. Most of the systemic vessels develop from a vast endothelial sheet that is connected to the endocardium of the developing heart (Coffin and Poole, 1988). Recent studies suggest that endothelial cells induce local mesenchyme to differentiate into smooth muscle (Folkman and D""Amore, 1996 and references within). Development of the intracardiac vessels contrasts this situation as endothelial and smooth muscle cell progenitors arise from the same epithelial structure (the proepicardial, organ; Manasek, 1968; Viragh and Challice, 1981; Mikawa and Gourdie, 1996; Dettman et al, 1998). These epithelial cells delaminate, migrate as single cells to distant sites within the heart and then differentiate. In addition, the intracardiac system is generated without connection to the systemic circulation (Poelmann et al, 1993; Viragh et al. 1993). Finally, a new bHLH molecule, capsulin, has been identified in the progenitors of smooth muscle of the intracardiac arterial system suggesting unique molecular regulation (Hidai et al, 1998; Lu et al, 1998). Thus, the ontogenesis of the intracardiac arterial system appears to be unique.
Organogenesis of the heart is a complex process where several independent yet interacting morphogenetic events proceed concurrently. Specific cellular process involved in heart development is not yet known. The prior art is deficient in the lack of a heart enriched cDNA library. Further, the prior art is deficient in the lack of identification and characterization of bves, a novel gene whose nucleic acid sequence is expressed in developing and adult heart and skeletal muscle. The present invention fulfills this long-standing need and desire in the art.
The present invention screens for novel gene products expressed during heart organogenesis and describes the identification and characterization of a novel gene product, Bves (Blood Vessel/Epicardial Substance). Bves is uniquely expressed in the proepicardial organ and a subset of its progeny gives rise to the vascular smooth muscle of the intracardiac arteries. Analysis of Bves expression reveals the migration and patterning of vascular smooth muscle in the heart and possible insights into the cellular regulation of smooth muscle differentiation during vasculogenesis.
The present invention discloses that bves is a novel mRNA expressed in the developing heart in chick (cbves), mouse (mbves) and human (hbves). cDNA sequences of cbves, mbves and hbves are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and deduced amino acid sequences in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively. bves is highly conserved between all three species at the amino acid level with 75% identity and 92% similarity.
In one embodiment of the present invention, there is provided a nucleic acid sequence encoding a Bves protein, wherein said sequence is selected from the group consisting of: (a) a nucleic acid which encodes a Bves protein; (b) a nucleic acid which is complementary to the nucleic acid of (a) encoding a Bves protein; and (c) a nucleic acid differing from the nucleic acids of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and (d) a nucleic acid of (a), (b) or (c) and which is either DNA or RNA. Specifically, the gene whose nucleic acid sequence is expressed in developing and adult heart and skeletal muscle cells in an organism is selected from the group consisting of chick, mouse and human. More specifically, the nucleic acid sequence comprises a cDNA sequence selected from the group consisting of SEQ ID Nos. 1-3, while the Bves protein has an amino acid sequence selected from the group consisting of SEQ ID Nos. 4-6.
In another embodiment of the present invention, there is provided an expression vector which expresses the desired nucleic acid when the vector is introduced into a cell.
In still another embodiment of the present invention, there is provided a host cell transfected with the vector which expresses a Bves protein. Preferably, the host cell can be a bacterial cell, an animal cell, a plant cell or an insect cell. Preferably, the bacterial cell is E. coli cell. More preferably, the host cell is the HT-1-bves engineered cell line.
In yet another embodiment of the present invention, there is provided an isolated and purified Bves protein coded for by a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid which encodes a Bves protein; (b) a nucleic acid complementary to the nucleic acid of (a) which encodes a Bves protein; and (c) a nucleic acid differing from the genes of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a Bves protein and (d) a nucleic acid of (a), (b) or (c) and which is either DNA or RNA. Preferably, the Bves protein has an amino acid sequence selected from the group consisting of SEQ ID Nos. 4-6.
In still yet another embodiment of the present invention, there is provided a recombinant protein having an amino acid sequence selected from the group consisting of SEQ ID Nos. 4-6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1-3. Preferably, the recombinant protein is an antigen.
In yet another embodiment of the present invention, there is provided a method of producing the recombinant protein, comprising the steps of obtaining a vector that comprises an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID Nos. 4-6 operatively linked to a promoter; transfecting the vector into a cell; and culturing the cell under conditions effective for expression of the expression region.
In still yet another embodiment of the present invention, there are provided antibodies immunoreactive with an amino acid sequence shown in SEQ ID No. 6.
The invention may also be described in certain embodiments as a method of detecting development of coronary vessels in an individual, comprising the steps of applying the antibody to the individual; and detecting the localization of the antibody. If the localization is detected in coronary vascular smooth muscle cells, the testing individual has developed coronary vessels.
The invention may also be described in another embodiment as a method of detecting a cardiovascular disease in an individual by applying the antibody and then detecting the localization of the antibody in the individual. If the localization is not detected in cardiovascular smooth muscle cells, the testing individual is suspected to have a cardiovascular disease. An example of such cardiovascular disease is atherosclerosis.
Similarly, the invention may also be described in still another embodiment as a method of detecting a skeletal muscle disease in an individual by detecting the immunolocalization of the antibody in skeletal muscle cells. Negative result indicates the possible existence of skeletal muscle disease in the testing individual. Examples of such skeletal muscle disease are muscular dystrophy and myonic dystonia.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.